Inflammation is a complex but essential innate immune defense mechanism. At the site of injury, a cascade of mediators, including cytokines (e.g. TNF, IL-1, IL-6, MCP-1, VEGF), eicosanoids, and reactive oxygen intermediates activate nearby inflammatory cells such as macrophages and granulocytes. The regulation of cytokines during inflammation is particularly critical as cytokine dysregulation is implicated in several diseases (e.g. type I diabetes, atherosclerosis, rheumatoid arthritis, Crohn's disease). In 1998 our laboratory discovered a new transcription factor, Lipopolysaccharide-Induced TNF-Alpha Factor (LITAF), which contributes to the transcriptional regulation of TNF and other proinflammatory cytokine genes. Separately, using serial analysis of gene expression a series of p53-induced genes were identified. LITAF was identified among these genes. Our preliminary data point to a specific site within the human LITAF promoter required for p53 protein-DNA binding. Transient transfection of either a p53 mutant DNA construct, called p53LFB164, or its corresponding synthetic peptide, reduced LITAF promoter activity and down-regulated LITAF expression. Furthermore, introduction of this peptide into human monocytes resulted in down-regulation of 9 cytokines including TNF-1. Since both TNF-1 and LITAF play important roles in human inflammatory diseases (i.e. Periodontal Disease, inflammatory bowel disease, Crohn's disease), clarification of the mechanism linking p53 and LITAF-mediated inflammatory processes may lead to novel therapeutic approaches for these or other inflammatory diseases. To address this important issue 2 aims are proposed: In Aim 1, deletions of LITAF promoter DNA (pMLFP) and a series of pcMp53 mutated DNAs will be constructed. To examine whether mouse LITAF promoter activity is regulated by mouse p53, p53-null cells will be co-transfected with pMLFP (or its deletion constructs) and pcMp53 (or its mutation constructs). Furthermore, EMSA and DNase I footprinting will be performed to determine the specific regions of p53 which interacts with the LITAF promoter. In Aim 2, we will fully characterize the mouse p53 short peptide that functions as an inhibitor to down-regulate LITAF-mediated in vitro and in vivo inflammatory cytokine production in response to lipopolysaccharide (LPS) stimulation. Mouse p53 short peptide will be synthesized and transfected or cotransfected with DNAs into p53-null cells for peptide-dependent promoter assays. In addition, peptides will be delivered into LPS-exposed cells, and the culture supernatants analyzed for cytokine expression. Finally, shortly after LPS injection, mice will be treated intravenously with p53 peptide and its effect on systemic LPS treatment will be fully analyzed. The data from these animal studies will be used to validate our in vitro observations and enable us to design pharmacotherapeutic approaches. Public Health Relevance: A common component in various chronic diseases and cancer development is the inflammatory process. The present work proposes to investigate a novel link between p53 and inflammation, and to test a potentially novel interventional approach to control p53-associated inflammatory processes. In addition, since loss or inactivation of p53 plays a key role in cancer development, understanding a link between p53 and the inflammatory process might shed light onto the established but incompletely understood connection between chronic inflammation and carcinogenesis.